There is an urgent need for effective therapeutic agents against human immunodeficiency virus 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). This project will result in the development of ribozymes that will selectively attack and destroy HIV RNA within an infected cell. Ribozymes are catalytic RNA molecules; many catalyze site-specific RNA cleavage reactions. The molecular basis for substrate selectivity is understood for some ribozymes. Therefore, it is possible to rationally alter the substrate specificity of a ribozyme by engineering changes within its substrate binding site using recombinant/synthetic DNA methods. The Specific Aims of this proposal are to (1) Develop and characterize ribozymes that selectively attack several HIV-1 RNA sequences, (2) Assemble and evaluate multivalent ribozymes against HIV-1 RNA, (3) Develop a bacteriophage system for analyzing and optimizing antiviral activity of ribozymes in vivo, (4) Use the bacteriophage system for surrogate genetic enrichment of optimized anti-HIV ribozymes, and (5) Evaluate the activity of resulting monovalent and multivalent anti-HIV ribozymes in T cell extracts. Ribozymes for this study will be derived from the self-cleaving "hairpin" minus strand of tobacco ringspot virus satellite ((-)sTRSV). In preliminary work, we have generated a hairpin ribozyme that selectively attacks HIV-1 pol sequences. Advantages of anti-HIV ribozymes are several: (i) Ribozymes can potentially inhibit HIV infection of cells and also inhibit HIV gene expression within infected cells, (ii) Ribozymes may be highly selective agents, (iii) Ribozymes act catalytically, so that a single molecule of ribozyme can potentially destroy many molecules of HIV RNA, and (iv) Ribozymes are not susceptible to pleiotropic drug resistance. This project will directly address major challenges in the development of optimal anti-HIV ribozymes---to achieve the optimal balance of sequence selectivity, catalytic activity and ribozyme stability under conditions encountered in vivo.